Imidacloprid Induces Neurotoxicity in Albino Male Rats by Inhibiting Acetylcholinesterase Activity, Altering Antioxidant Status, and Primary DNA Damage

Imidacloprid (IMI) is a neonicotinoid insecticide used worldwide, either alone or in combination with other pesticides. The goal of this study was to assess the effects of IMI on the central nervous system of rats and its mechanism of oxidative stress-induced DNA damage by oxidant/antioxidant parameters. Fifteen male rats, divided into three groups, were used: the first group received 5 ml/kg body weight corn oil as a control, the second received a high oral dose of IMI (45 mg/kg body weight), while the third received a low dose (22 mg/kg body weight). After 28 days, acetylcholinesterase (AChE) activity, oxidative stress markers, histopathological alterations, and DNA damage were examined in the brains of these rats. The AChE activities decreased significantly after IMI exposure, reaching 2.45 and 2.75 nmol/min/mg protein in high dose and low dose, respectively, compared to the control group (3.75 nmol/g tissues), while the concentration of malondialdehyde MDA increased significantly (29.28 and 23.92 nmol/g tissues) vs. the control group (19.28 nmol/g tissues). The antioxidant status parameters such as reduced glutathione (GSH) content was 13.77 and 17.63 nmol/g, catalase (CAT) activity was 22.56 and 26.65 µmol/min/g, and superoxide dismutase (SOD) activity was 6.66 and 7.23 µmol/min/g in both doses against the control group (21.37 nmol/g, 30.67 µmol/min/g, 11.76 µmol/min/g), respectively, and histopathological changes in the brain tissues were observed. More in vivo research using epigenetic methods is needed to determine the ability of IMI and its metabolites to cause neurotoxicity and DNA lesions in mammalian brains.


Introduction
Neonicotinoids, which frst appeared in the late 1980s, now constitute one-third of the global pesticide market and are rapidly replacing organophosphates, carbamates, and pyrethroids [1].Nicotine is a part of their structure, and they specifcally act on nicotinic acetylcholine receptors [2].Imidacloprid (IMI) is an agonist nicotinic acetylcholine receptor that is very efective against a variety of sucking insects [3,4].Its action causes an in acetylcholine, which paralyzes the insect and eventually kills it [3].Inhibition of the enzyme acetylcholinesterase (AChE) was one of the frst biomarkers identifed for human exposure to environmental pesticides.Guerra et al. found a statistically signifcant decrease in zebrafsh brain AChE activity after being exposed to 15 and 45 µg/L IMI for 96 hours [5].On the other hand, Sevim et al. discovered no signifcant diferences in AChE levels between IMI-exposed and control L-929 fbroblasts [6].
Oxidative stress and lipid peroxidation (LPO) are considered good biomarkers for assessing insecticide-induced hazardous efects in numerous organisms [7][8][9].Oxidative damage harms biological systems by contributing to lipid, DNA, and protein damage, leading to cell death.Numerous studies have shown that IMI causes oxidative stress and lipid peroxidation in mammals and cell lines [10,11].Orally exposing female rats to 20 mg/kg IMI resulted in a signifcant elevation of malondialdehyde (MDA) levels and a decrease of redox parameters such as reduced glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) in their livers, kidneys, and brains [12].Furthermore, IMI disrupts compound ion channels or neurotransmission, leading to irreversible neurotoxicity in mammals which may eventually devolve into chronic neurodegenerative disorder.
Humans are primarily exposed to IMI through drinking water and food.Concerningly, IMI can cause carcinogenic and mutagenic efects in both animals and humans [13].Te frst stage of genotoxicity is DNA damage, which disrupts biological structures and functions, and causes reproductive and carcinogenic problems [14].DNA oxidation is a useful marker for determining mutagenicity mechanisms [15].IMI's mutagenic potential has been demonstrated in vitro and in vivo using genomic endpoints, for instance, the micronucleus test, chromosomal aberrations, the comet assay, and DNA damage [16][17][18].Moreover, exposure occurs in the nutrition through imported agricultural goods, till now [19].Te aim of this research was to discover the mechanism of neurotoxicity by which IMI afects cholinesterase activity, antioxidant status biomarkers, and primary DNA damage in rat brains after a 28-day oral dosage.

Experimental Design.
Te IMI doses were chosen based on the documented median lethal dose value: 450 mg/kg body weight [20].Te time period of the experiments was varied according to the results of the subacute study conducted by Test No. 407 [21].IMI was dissolved in corn oil and administered orally daily for 4 weeks.Te animals were divided randomly into three groups (n � 5 per group) and gavage-treated for 28 days at 45 mg/kg B.W. IMI for high dose and 22.5 mg/kg B.W. IMI for low dose whilst the control group received 5 ml/kg B.W. with corn oil as vehicle.

Euthanasia and Sample
Collection.After 28 days, the animals were anaesthetized prior to cervical dislocation with intraperitoneal injections of ketamine (90 mg/kg B.W.) and xylazine (5 mg/kg B.W.) [22].Brain samples were quickly extracted, rinsed in ice-cold saline, homogenized, and used to determine biochemical parameters.Te brain homogenates were centrifuged at 14000 rpm for 20 minutes at 4 °C (MSE Super-Minor centrifuge, England), and the supernatants were collected to measure LPO levels (MDA), GSH contents, antioxidant enzyme activities (CAT and SOD), and AChE activity using the V-670 UV/VIS/NIR spectrophotometer (190-2700 nm wavelength).Brian tissue samples were also dissected for examining DNA fragmentation and their histopathology.

Acetylcholinesterase Activity.
AChE activity was evaluated using a previously described method by the authors in [23].Te reaction mixture, containing 2.6 ml of sodium phosphate bufer (0.1 M, pH 7.5), 0.15 ml of dithiobis (2-nitrobenzoic acid) (DTNB, 10 mM, pH 7.0, containing 3 mg of NaHCO 3 per 8 mg of DTNB), and 0.1 g of the supernatant, was kept at room temperature for 10 min.Te reaction was started by adding 0.15 ml of acetylthiocholine iodide (12.5 mM), and the absorbance change per minute was measured at 412 nm for 4 min.Specifc activity is expressed in nmol•min −1 •mg −1 protein.

Lipid Peroxidation
Level.MDA, a byproduct of LPO, is commonly detected by the thiobarbituric acid reactive substances (TBARS) assay because it reacts with TBARS to produce a fuorescent product [24].Tiobarbituric acid (TBA) reacts with MDA in an acidic medium for 30 min at 95 °C to form a TBAR product, which can be measured at 534 nm.MDA levels are expressed in nmol g −1 tissue.

Oxidative Stress Markers.
GSH content was assessed by the method described in [25], CAT activity was estimated using a previously described method [26], and SOD activity was measured using the autoxidation and illumination of pyrogallol at 440 nm for 3 min 40, according to Marklund and Marklund [27].
2.8.Total DNA Fragmentation.DNA fragmentation was qualitatively assessed by agarose gel electrophoresis through a DNA laddering assay [28,29].To clarify, the brain tissue was homogenised in a hypotonic lysis bufer, the lysates were centrifuged for 10 minutes, and the supernatants containing small DNA fragments were separated from the intact DNA pellets.Te DNA was resuspended in Tris-ethylenediaminete traacetic acid (Tris-EDTA) bufer for electrophoretic analysis on a 2% agarose gel.Under ultraviolet light, the gel was stained with ethidium bromide and observed.
2.9.Histopathological Examination.Brain cells were collected from all groups, immersed in 10% formol-saline for 24 h, embedded in parafn wax, sectioned at 4 mm, and stained with hematoxylin and eosin.Te slides were examined under a microscope to assess pathomorphological alterations in the brain tissues [30].

Statistical Analysis.
Te data are displayed as mean-± standard deviation (M ± SD).Te one-way ANOVA (SPSS, SPSS Inc., Chicago, IL, USA) version 17 and GraphPad Prism, version 8 software (San Diego, CA), compared the groups.A p value of <0.05 is recorded.

Results and Discussion
3.1.Acetylcholinesterase Activity.Te nicotinergic neurons in the brain are inactivated by IMI, a chloronicotinyl insecticide used systemically [31,32].As shown in Figure 1 and Table 1, all investigated doses of IMI signifcantly reduced AChE activity in rat brains after 28 days, compared with the control (p < 0.05), eventually leading to neurotoxicity.IMI binds, either with high afnity or partially, to specifc subsites on nicotinic acetylcholine receptors (nAChRs), stimulating them and inducing neurotoxicity [19,33,34].Tis continuous stimulation afects both nervous function and AChE activity [35].Kimura-Kuroda et al. showed that exposing a Sprague-Dawley rat cerebellar cell line to IMIinduced conformational changes in the Ach receptors and enhanced cellular Ca 2+ uptake via α7 nAChRs [32].Increased total cholinesterase activity, which reduces AChassisted IMI binding, may be an adaptive response to oxidative stress caused by increased Ca 2+ uptake [36].Like our results, Topal et al. reported decreased AChE activity in the brains of fsh exposed to IMI [37].

Lipid Peroxidation.
Increased free radicals generated during IMI exposure, combined with the decreased capacity of the cell to scavenge them, may be responsible for free radical-induced membrane lipid peroxidation (MDA) in rat tissues [10].Figure 2 and Table 1 show that MDA signifcantly increased in brain tissues following both the oral doses of IMI, compared with the control.A high concentration of polyunsaturated fatty acids in biological systems, combined with a low antioxidant capacity, makes them more susceptible to LPO and oxidative stress [38].Furthermore, IMI metabolites, such as desnitro metabolites, and nitromethylene analogs are more dangerous to mammals [19,31,39].IMI metabolites might be sources of free radicals that induce abundant amounts of MDA.Numerous fndings have suggested that IMI exposure is associated with LPO in rodent livers and kidneys [12,40].

Oxidative Stress Biomarkers. Pesticide concentrations and structures may alter the efciency of the GSH redox cycle.
Furthermore, enzymes such as SOD and CAT neutralize the reactive oxygen species (ROSs) produced during pesticide exposure [10,41].GSH and the vitamins C and E have been shown to protect cells and tissues from xenobiotic oxidative stress [42].Several studies have shown that IMI has oxidative and neurotoxic potential in mammals [32,39,43].
Figures 3-5, and Table 1 show that all doses of IMI led to a signifcant decrease in GSH, CAT, and SOD levels compared with the control (p < 0.05).IMI's metabolism is fast and metabolite accumulation in tissues may result in an excess of free radicals/ROS, which could damage mitochondrial membrane structures, having caused permeability changes as evidenced by increased LPO and GSH depletion [44].Mahajana et al. showed that IMI exposure reduced antioxidant enzymes (SOD and CAT) responsible for scavenging superoxides, peroxides, and hydroxides generated during toxicant-induced oxidative stress in the kidney tissue [10].
3.4.Total DNA Fragments.DNA damage can disrupt biological pathways, resulting in a genotoxic disorder linked with reproductive and carcinogenic diseases [45].IMI's mutagenic potential has been demonstrated in in vitro and in vivo systems using cytogenetic endpoints, such as the micronucleus test, the comet assay, and the DNA fragmentation assay [13].Figure 6 shows that brain cells frequently contained fragmented DNA, observed using gel electrophoresis, and these fragmentations were used as a damage characteristic.DNA, isolated from the brains of rats exposed to IMI at doses of 45 mg/kg and 22 mg/kg body weight, had degraded into oligonucleotide fragments, forming a clear laddering pattern when resolved by electrophoresis.Te higher dose caused more DNA fragmentation, yielding four bands (790.12,627.72, 436.2, and 258.47 bp) compared with control, while the lower dose yielded three bands (810.59,622.61, and 439.91 bp).ROS levels increased with increasing IMI concentrations, resulting in more DNA damage [18,46,47].Change in glutathione status due to toxic agents causes oxidative stress, which leads to mammalian genotoxicity [48].IMI is an alkylating material that can cause clastogenesis by damaging cellular DNA.Te presence of an electronegative group in the nitroguanidine moiety of neonicotinoids may link IMI to Journal of Toxicology DNA, inducing the formation of breaks [2].IMI-induced DNA damage could also be attributed to direct reactions with the pesticide or their metabolites, which destabilize the DNA structure and cause breaks [49].

Histopathological Findings.
Te histopathology of the cerebral cortex, hippocampus, striatum, and the cerebellum of control group rats was unaltered, thus showing a normal histological neuronal structure (Figure 7).Rats exposed to the lower dose of IMI showed nuclear pyknosis and degeneration in the neurons of the cerebral cortex, fascia dentata, and the hilus, with intracellular edema in the

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Journal of Toxicology striatum, and necrosis in the granular layer of the cerebellum (Figure 8).Similar observations were made in rats administered the higher dose of IMI, along with focal eosinophilic plagues in the striatum (Figure 9).Tese histopathological fndings are in line with the results of Abd-Elhakim et al., who found varying degrees of neuronal degeneration in the brains of male rats exposed to 1 mg/kg body weight IMI per day [50].In addition, exposure to IMI for 28 days induced necrosis of Purkinje cells with loss of dendrites and granules in the granular layer of the cerebellum in female rats [43].
Te brain of IMI-treated mice 28 (for 90 days) revealed focal neuronal degeneration, hemorrhages, necrosis, and congestion of cerebral blood vessels, besides congestion and degeneration in areas of the hippocampus [51].IMI was detectable and caused DNA damage in the brains of male rats treated orally with it for 28 days (2.25 mg/kg body weight/day) [18].Degenerative changes in Purkinje cells of the cerebrum have been recorded in rats exposed to high doses of IMI [52].Also, brain sections of rats treated with IMI displayed perivascular hemorrhage and nuclear

Conclusion
Te subacute IMI exposure can cause toxic efects, which can lead to a variety of neurodegenerative diseases, depending on the dose which its outcomes AChE inhibition; changes in antioxidant status caused by increased lipid peroxidation and a decrease in GSH, CAT, and SOD.In addition, high and low doses induced genotoxicity by DNA fragmentation.Te results show that IMI can decrease new neurons, through deterioration in brain cells by histopathological examination and cell membrane via increment of lipid peroxidation.Our data suggest that IMI induces oxidative stress which produces ROS leading to abnormal total DNA, moreover inhibition of acetylcholinesterase activity in rat brain.All of the abovementioned biological events have been suggested to be possible mechanisms for IMI neurotoxicity.Tese results provide a possible theoretical basis for evaluating the harm of IMI to mammals and environment.Tus, we can conclude from our study that the nervous system of mammals, including humans, is more sensitive to excessive consumption of IMI in the environment as a replacement for traditional insecticides, and that much more research is needed to prove how IMI and its metabolites promote oxidative stress, which causes alterations in the mRNA levels of genes that encode antioxidant proteins.

Figure 1 :
Figure 1: Acetylcholinesterase activity in rat brain tissues after 28 days of oral IMI exposure.Values are expressed as the mean ± SD expressed as nmol of acetylthiocholine hydrolyzed per minute per mg protein.45 mg/kg B.W., and 22.5 groups vs. control p < 0.05 three groups (n � 5).

Figure 2 :Figure 3 :
Figure 2: Malondialdehyde levels in rat brain tissues after 28 days of oral IMI exposure.Values are expressed as the mean ± SD expressed as nmol of MDA per g protein.45 mg/kg B.W., and 22.5 groups vs. control p < 0.05 three groups (n � 5).

Figure 4 :
Figure 4: Catalase levels in rat brain tissues after 28 days of oral IMI exposure.Values are expressed as the mean ± SD expressed as units of CAT per g protein.45 mg/kg B.W., and 22.5 groups vs. control p < 0.05 three groups (n � 5).

Figure 5 :
Figure 5: Superoxide dismutase levels in rat brain tissues after 28 days of oral IMI exposure.Values are expressed as the mean ± SD expressed as units of SOD per g protein.45 mg/kg B.W., and 22.5 groups vs. control p < 0.05 three groups (n � 5).

Figure 6 :
Figure 6: Agarose gel electrophoresis was used to evaluate the fragmentation of rat brain DNA in all experimental groups.Ladder DNA marker, DNA patterns of the control group, the treated group (low dose), and the treated group (high dose).

Figure 7 :
Figure 7: Displayed a photomicrograph of the cerebral cortex, subiculum, fasci dentata and hilus, straitum, and cerebellum in rat brain tissues for histopathological extermination in a control group.

Figure 8 :
Figure 8: Displayed a photomicrograph of the cerebral cortex, subiculum, fasci dentata and hilus, straitum, and cerebellum in rat brain tissues for histopathological changes in a low dose group.

Figure 9 :
Figure 9: Displayed a photomicrograph of the cerebral cortex, subiculum, fasci dentata and hilus, straitum, and cerebellum in rat brain tissues for histopathological changes in a high dose group.

Table 1 :
Te efects of sublethal Imidacloprid doses on antioxidant parameters, lipid peroxidation, and neural biomarker (AChE) in rat brain tissues.Data are expressed as mean ± SD (n � 5) a Statistical signifcance between imidacloprid treatment groups versus control group (the one-way ANOVA, p < 0.05).